| β-Cyclodextrin glycosyltransferase(β-CGTase,EC2.4.1.19)is one unique member of a-amylase family.β-CGTase is a multifunctional enzyme. It can catalyze transglycosylation reactions:disproportionation, cyclization, coupling and hydrolysis reaction. Cyclization is (3-CGTase effect on substrate to convert starch intoβ-Cyclodextrin.β-Cyclodextrin have a hydrophilic outer surfase and nonpolar hole, it can embed many organic matter and some inorganic, as quality improver, stabilizer, drug carrier and the absorbent, etc.and leading to improvement of physical and chemical properties of the compounds. Therefore,β-Cyclodextrin products have been widely used in pharmaceutical,food, agriculture,chemical, cosmetic and environmental protection,et al.which make it very important to study the Production.Thermophiles are abundant in the nature environment:they are thermostable and thertno-philie, and they also have excenlant degradation capability and high metabolie Effiency, and obtain easily high purity, no miscellaneous bacteria survive, which leads to the wide use of their enzymes in some industries. However, the current research of thermophiles is inadequate.Inorder to better use the thermophiles resource and facilitate broader utilization of these resources in more industries, more therinophilies with high degradation capability are needed,thermostable strain HY15,selected and conserved in our lab,it is aim of the high yield ofβ-CGTase.In this study,β-CGTase production by this strain HY15 was systematically investigated for the purpose of high enzymatic yield.The biochemical characteristics and dynamic properties of fermentation were explored. The fermentation and fermentation conditions on the influence of enzyme production were studied systematic. In order to need the requirement of industrialization production.Still further experimental research on 5.0 L automatic fermentation tank, and study on purification methods of enzymes and enzyme kinetics properties. For this enzyme can provide theoretical basis and technical support for industrial production. The summarize research contents are as follows:1. The optimal fermentation conditions forβ-CGTase prodtion.Through the single factor and shaking flasks experiment.Molasses and Corn starch was selected as the best carbon source for theβ-CGTase production by strain HY15. Corn steep liquor and yeast were also selected as the suitable nitrogen sources.β--CGTase yield and cell growth were significantly promoted by the addition of cations such as Mg2+,PO43-. The combined addition of 10 mmol/L Mg2+,120 mmol/L PO43-. The optimal initial pH.temperature and medium volume in 250 mL shaking flasks for enzyme fermentation were 7.0,60℃and 50 mL,The appropriate inoculum size was 3%,the rotation speed of shaker was 200r/min. Fermentation time was 48h.On the base of single factor experiment.The Plackett-Burman design was used to evaluate the effects of ten variables which could affect theβ-CGTase production.On the basis of analyzing the steepest ascent experiment was adopted to determine the optinal region of the medium composition. Subsequently,response surface methodology was employed to determine the maximumβ-CGTase production at the optimum concentration of key factors,in which,a quadratic polynomial model for P-CGTase yield was established.The optimal fermentation conditions for P-CGTase prodction were determined.The results is theβ-CGTase production was found to correlate to the seven parameters:molasses,corn starch,K2HPO4, MgSO4, corn steep liquor and yeast extract,pH was found to be the most important factors.The optimal values of the five parameters were determined as 22.45g/L molasses and corn starch,0.97g/L K2HPO4, MgSO40.18g/L,21.82g/L corn steep liquor and yeast extract, pH 6.98. In the optimum conditions.Theβ-CGTase production was 1401.81U/mL,which shows the results are reasonable.2. The optimal fermentation conditions of 5.0 L scale fermentor. The fermentation rule of strain HY15 were studied in a 5.0L scale fermentor for enzyme activity, total sugar concentration,cell growth,pH. The influence of various agitation speeds and dissolved oxygen (DO) levels onβ-CGTase production and cell growth. When the agitation speed was controlled at 300 r/min and DO was controlled at 40%,the temperature was controlled at 60℃, the pH was controlled at 7.0, theβ-CGTase yield reached 2411U/mL,OD600 reached 1.88. Fermentation after 8h,flow fed 20g/L corn steep liquor and molasses 30g/L,theβ-CGTase yield and OD600 were both increased, theβ-CGTase yield reached 2700U/mL,OD600 reached 1.92.which was increased 3.3 times of primitiveβ-CGTase 817 U/mL3. Purification of P-CGTase. The specific activity of theβ-CGTase Produced by strain HY15 in liquid cultures was enhanced by16 folds after a series of purification steps including ammonium sulfate precipitation,sepharoseCL-6B chromatography.Polyacrylamide gel electro-phoresis (SDS-PAGE) analysis indicates that the purified enzyme was homogeneous with its subunit molecular mass being estimated as 69kDa in SDS-PAGE.4. Characteristics of the purifiedβ-CGTase. The optimal conditions for reaction of the P-CGTase produced by strain HY15 was pH6 at 60℃.The enzyme was stable in a range of pH6.0-9.0 at the temperature below 80℃. The enzyme was inactivated at the presence of Al3+,Ag+, Fe2+,Cu2+ at the level of lmmol/L.Whereas it was activated at the same level of Ca2+,Mn2+, Mg2+.However,Zn2+,K+,Cr3+had insignificant effect on the activity of the P-CGTase concenred.The km value for its reaction on soluble starch was 0.379 mg/mL,the Vmax value was 13.24mg/min. The km value for its reaction on potato starch was 0.476 mg/mL,the Vmax value was 6.36mg/min. The km value for its reaction on corn starch was 0.3125mg/mL,the Vmax value was 13.29mg/min.Its explained the affinity is strongest of the potato starch andβ-CGTase of strain HY15.Based on HPLC analysis, hydrolysis product of the P-CGTase reacted on corn starch were researched.The starch can transformβ-CD.In the process ofβ-CGTase reaction on the corn starch.The sugar content and the time display the relevance of double exponential. It have varying effects which temperature, pH, enzyme concentration, starch concentration reacted on the process.In all,the thermophilic bacteria strain HY15 can produce heat-stable,great affinityβ-CGTase, which is significant potential for the application and development of P-CD...
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